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Identification of platelet function defects by multi-parameter assessment of thrombus formation.

de Witt SM, Swieringa F, Cavill R, Lamers MM, van Kruchten R, Mastenbroek T, Baaten C, Coort S, Pugh N, Schulz A, Scharrer I, Jurk K, Zieger B, Clemetson KJ, Farndale RW, Heemskerk JW, Cosemans JM - Nat Commun (2014)

Bottom Line: Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3.Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome.We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands.

ABSTRACT
Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

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Different types of thrombi formed on various microspots.Blood from control subjects was perfused over microspots with indicated coating for 3.5 min at 1,600 s−1. Shown are representative microscopic images (>5 donors), from left to right: phase-contrast images of adhered platelets (i); fluorescence images of platelets binding FITC-labelled anti-fibrinogen mAb (ii), FITC-labelled anti-CD62P mAb (iii) or AF647-annexin A5 (iv); further, z-stacks from aggregates of DiOC6-labelled platelets (v). Bar, 10 μm. Images from typical surfaces are given (for all 52 surfaces, see Supplementary Fig. 1).
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f3: Different types of thrombi formed on various microspots.Blood from control subjects was perfused over microspots with indicated coating for 3.5 min at 1,600 s−1. Shown are representative microscopic images (>5 donors), from left to right: phase-contrast images of adhered platelets (i); fluorescence images of platelets binding FITC-labelled anti-fibrinogen mAb (ii), FITC-labelled anti-CD62P mAb (iii) or AF647-annexin A5 (iv); further, z-stacks from aggregates of DiOC6-labelled platelets (v). Bar, 10 μm. Images from typical surfaces are given (for all 52 surfaces, see Supplementary Fig. 1).

Mentions: For a comparative analysis, whole blood from healthy subjects was perfused over microspots (three per run) containing the 52 combinations of substrate proteins and peptides at a high (arterial) wall shear rate of 1,600 s−1. Thrombus formation on all surfaces was measured using standardized microscopic procedures (see Methods). Stable platelet adhesion of DiOC6-labelled platelets was evaluated from sequential fluorescence images captured in real time during blood flow. Thrombus volume was assessed from z-stacks of confocal images of DiOC6 fluorescence at end stage. End stage, phase-contrast images were captured to determine overall platelet deposition (surface area coverage) and platelet aggregate size. Activation of the platelets per surface was resolved from confocal fluorescence images after labelling with fluorescein isothiocyanate (FITC)-labelled anti-fibrinogen monoclonal antibody (mAb) (fibrinogen binding, integrin αIIbβ3 activation), FITC-anti-CD62P mAb (P-selectin expression) or AF647-annexin A5 (procoagulant activity). As illustrated in Fig. 3, platelet adhesion, aggregation and activation markedly differed between the various microspots. No platelets adhered to spots coated with BSA (negative control), while single platelets with low activation state adhered to coated fibrinogen. Small aggregates of platelets with activated integrin αIIbβ3 and surface-expressed P-selectin formed on coated vWF or collagen III. Large platelet aggregates staining for all three activation markers formed on collagen I microspots, as expected39.


Identification of platelet function defects by multi-parameter assessment of thrombus formation.

de Witt SM, Swieringa F, Cavill R, Lamers MM, van Kruchten R, Mastenbroek T, Baaten C, Coort S, Pugh N, Schulz A, Scharrer I, Jurk K, Zieger B, Clemetson KJ, Farndale RW, Heemskerk JW, Cosemans JM - Nat Commun (2014)

Different types of thrombi formed on various microspots.Blood from control subjects was perfused over microspots with indicated coating for 3.5 min at 1,600 s−1. Shown are representative microscopic images (>5 donors), from left to right: phase-contrast images of adhered platelets (i); fluorescence images of platelets binding FITC-labelled anti-fibrinogen mAb (ii), FITC-labelled anti-CD62P mAb (iii) or AF647-annexin A5 (iv); further, z-stacks from aggregates of DiOC6-labelled platelets (v). Bar, 10 μm. Images from typical surfaces are given (for all 52 surfaces, see Supplementary Fig. 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109023&req=5

f3: Different types of thrombi formed on various microspots.Blood from control subjects was perfused over microspots with indicated coating for 3.5 min at 1,600 s−1. Shown are representative microscopic images (>5 donors), from left to right: phase-contrast images of adhered platelets (i); fluorescence images of platelets binding FITC-labelled anti-fibrinogen mAb (ii), FITC-labelled anti-CD62P mAb (iii) or AF647-annexin A5 (iv); further, z-stacks from aggregates of DiOC6-labelled platelets (v). Bar, 10 μm. Images from typical surfaces are given (for all 52 surfaces, see Supplementary Fig. 1).
Mentions: For a comparative analysis, whole blood from healthy subjects was perfused over microspots (three per run) containing the 52 combinations of substrate proteins and peptides at a high (arterial) wall shear rate of 1,600 s−1. Thrombus formation on all surfaces was measured using standardized microscopic procedures (see Methods). Stable platelet adhesion of DiOC6-labelled platelets was evaluated from sequential fluorescence images captured in real time during blood flow. Thrombus volume was assessed from z-stacks of confocal images of DiOC6 fluorescence at end stage. End stage, phase-contrast images were captured to determine overall platelet deposition (surface area coverage) and platelet aggregate size. Activation of the platelets per surface was resolved from confocal fluorescence images after labelling with fluorescein isothiocyanate (FITC)-labelled anti-fibrinogen monoclonal antibody (mAb) (fibrinogen binding, integrin αIIbβ3 activation), FITC-anti-CD62P mAb (P-selectin expression) or AF647-annexin A5 (procoagulant activity). As illustrated in Fig. 3, platelet adhesion, aggregation and activation markedly differed between the various microspots. No platelets adhered to spots coated with BSA (negative control), while single platelets with low activation state adhered to coated fibrinogen. Small aggregates of platelets with activated integrin αIIbβ3 and surface-expressed P-selectin formed on coated vWF or collagen III. Large platelet aggregates staining for all three activation markers formed on collagen I microspots, as expected39.

Bottom Line: Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3.Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome.We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands.

ABSTRACT
Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

Show MeSH
Related in: MedlinePlus